The physical and chemical factors that allow polynucleotides to perform their functions in the cell have been studied for several decades. Recent advances in the synthesis and manipulation of polynucleotides have allowed this field to move ahead especially rapidly during the past fifteen years. One of the most common chemical approaches to the study of interactions involving biomolecules has been the use of nucleoside base analogs in which functional groups are added, deleted, blocked, or rearranged.
Such nucleoside analogs may be useful as in providing specific alterations to reaction kinetics; properties to oligonucleotide probes for diagnostic applications; to alter the properties of antisense RNA and RNAi; and in the synthesis and purification of oligonucleotides. Among the uses of oligonucleotides are methods of inhibiting gene expression with antisense oligonucleotides complementary to a specific target messenger RNA (mRNA) sequences. Oligonucleotides also have found use in diagnostic tests performed using biological fluids, tissues, intact cells or isolated cellular components. For diagnostics, oligonucleotides and oligonucleotide analogs can be used in cell free systems, in vitro, ex vivo or in vivo.
Oligonucleotides and nucleosides are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of such other biological molecules. For example, oligonucleotides serve as primers in the reactions associated with polymerase chain reaction (PCR), which reactions are now widely used in forensics, paleontology, evolutionary studies and genetic counseling, to name just a few.
The use of antisense oligonucleotides to knock down gene expression is of great interest. In many cases, gene knockdowns with oligonucleotides have been achieved primarily through the injection of antisense molecules, after which the targeted RNAs are silenced for a period of up to several days. However, this does not permit conditional gene silencing. One approach for overcoming temporal and/or spatial limitations on knockdowns utilizes “caged” molecules that are photoactivatable, thereby providing for temporal or tissue specificity. Improved methods and compositions for conjugated linkers to oligonucleotides are of interest for a variety of applications.
Among widely used antisense molecules are morpholino oligonucleotides, which contain a six-membered morpholine ring in place of a ribose sugar, and a phosphorodiamidate backbone. Morpholinos are often used as a research tool for reverse genetics by knocking down gene function, and are also in development as pharmaceutical therapeutics targeted against pathogenic organisms and for amelioration of genetic diseases. Because of their synthetic backbone, morpholinos are not recognized by cellular proteins, and so are not degraded by nucleases in cells or in serum. Activities of morpholinos against a variety of targets, including miRNA, mRNA, and ribozymes suggest that they can be used as a general-purpose tool for blocking interactions of proteins or nucleic acids with mRNA.
Linker compounds that allow photoactivation of molecules are of great interest for a number of purposes, including photoactivation of drugs, antisense oligonucleotides, and the like. The present invention provides linkers and compositions derived therefrom for such purposes.